1. Field of the Invention
The present invention relates to a method of using, in a reversed phase chromatography, a stationary phase comprising an alkyl group as a main component thereof, with a mobile phase comprising water as a main component thereof, and also relates to a reversed phase chromatograph apparatus comprising a stationary phase comprising an alkyl group as a main component thereof and a mobile phase comprising water as a main component thereof.
2. Related Art Statement
There are known a reversed phase chromatography in which water is used as a main component of a mobile phase as a solvent for dissolving a sample including a water soluble compound to be separated, and a non-polar compound is used as a main component of a liquid or solid stationary phase supported by a solid support filled in a column, and a reversed phase chromatograph apparatus comprising the mobile phase and the stationary phase. In view of hydrophobicity and compound-retention capacity, an alkyl group having 8 carbon atoms (octyl group) to 18 carbon atoms (octadecyl group; hereinafter, referred to the "ODS") has been widely used as the stationary phase.
However, in the case where an alkyl group having 8 to 18 carbon atoms is used as the stationary phase, if 100% of water is used as the mobile phase for the purpose of separating a water soluble compound, a time-wise change occurs that the sample-retention or compound-retention time between the time when a sample is injected into the column and the time when the compound is detected under the column gradually decreases. FIG. 1 shows three chromatograms for illustrating a time-wise change that was observed when an identical sample is used at three different times, each for separating five sorts of compounds. The ODS (octadecyl group) is used as the stationary phase, and 100% of water is used as the mobile phase. The three chromatograms of FIG. 1 prove that as time passes, the compound-retention time of the column, i.e., the stationary phase gradually decreases, which leads to an incomplete separation of the compound. Thus, in the case where 100% of water is used as the mobile phase, the ODS cannot be used as the stationary phase. It is thought that the above time-wise change would result from the "slipping" phenomenon that as the 100% water mobile phase iteratively passes through the column, the carbon chains (ligands) gradually lie on their sides, as illustrated in FIG. 2, and the interaction between the stationary phase and the mobile phase gradually decreases.
For preventing the above time-wise change, it has been proposed to use, as the stationary phase, an alkyl group having a smaller number of carbon atoms that is physically thought to less likely lie on its side. FIG. 3 shows three chromatograms in which trimethylsilyl group is used as the stationary phase, 100% water is used as the mobile phase, and the same sample as used in those shown in FIG. 1 is used at three different times. The three chromatograms of FIG. 3 prove that the compound-retention time of trimethylsilyl group used as the stationary phase does not decrease even though it may be used for a long time. It has also been proved that in the case where phenyl group is used as the stationary phase, its compound-retention time does not decrease. Those proofs also support that the above time-wise decrease in the compound-retention time of the alkyl group having 8 to 18 carbon atoms used as the stationary phase results from the "slipping" phenomenon.
However, in the case where an alkyl group having a small number of carbon atoms is used as the stationary phase, the stationary phase cannot completely separate different isomers from each other, because the alkyl group has only a poor steric selectivity. In addition, since the alkyl group has only a low hydrophobicity, it cannot sufficiently separate certain compounds from samples. Meanwhile, a stationary phase in which ODS is bonded at a lower density to the solid support and trimethylsilyl group is additionally bonded with the solid support, has been developed. This stationary phase enjoys the advantage of excellent compound-retention capacity of ODS and the advantage of trimethylsilyl group that a solution including water as a main component can be used as the mobile phase. This stationary phase has solved the problem of the time-wise decrease of compound-retention time to some degree, but not a satisfactory degree. In addition, since the amount of trimethylsilyl group is more, i.e., the amount of long alkyl group is less, the hydrophobicity of this stationary phase is lower than that of the prior stationary phase in which only the long alkyl group is used. Thus, this stationary phase suffers from the problems of low compound-retention capacity, insufficient compound-separate capability, and low steric selectivity.